This invention relates to the fields of pharmaceutical and organic chemistry and provides a cryptophycin compound useful in the treatment of cancer.
Neoplastic diseases, characterized by the proliferation of cells not subject to the normal control of cell growth, are a major cause of death in humans and other mammals. Clinical experience in cancer chemotherapy has demonstrated that new and more effective drugs are desirable to treat these diseases. Such clinical experience has also demonstrated that drugs which disrupt the microtubule system of the cytoskeleton can be effective in inhibiting the proliferation of neoplastic cells.
The microtubule system of eukaryotic cells is a major component of the cytoskeleton and is in a state of dynamic assembly and disassembly. Heterodimers of tubulin are polymerized to form microtubules. Microtubules play a key role in the regulation of cell architecture, metabolism, and division. The dynamic state of microtubules is critical to their normal function. With respect to cell division, tubulin is polymerized into microtubules that form the mitotic spindle. The microtubules are then depolymerized when the mitotic spindle""s use has been fulfilled. Accordingly, agents that disrupt the polymerization or depolymerization of microtubules, and thereby inhibit mitosis, comprise some of the most effective cancer chemotherapeutic agents in clinical use.
The cryptophycins are novel macrolides first isolated from blue-green algae (Nostoc sp. Strain GSV224) which have been shown to be potent tumor selective cytotoxins in vivo. Cryptophycins have been synthesized by a convergent method in which four fragments, Fragment A, Fragment B, Fragment C, and Fragment D (Golakati, et al., Journal of the American Chemical Society, 117(49), 12031 (1995)) are coupled together to form the final product (U.S. Pat. No. 6,013,626). Such methods have been used to prepare the original cryptophycins, isolated from Nostoc sp., as well as new cryptophycins.
Certain cryptophycin compounds, and methods for their preparation, are known in the literature. U.S. Pat. No. 4,845,085 and U.S. Pat. No. 4,845,086, for example, report novel semi-synthetic compounds formed by converting the epoxide of a particular cryptophycin to a carbon-carbon double bond. These compounds have potential as agents for mycotic infections.
U.S. Pat. No. 4,868,208 reports novel semi-synthetic antifungal compounds formed by subjecting a cryptophycin compound to hydrolysis or alcoholysis.
WO 95/17093, WO 96/39829, and WO 96/40184 report novel Cryptophycin compounds for inhibiting the proliferation of hyperproliferative cells.
WO 97/07798 reports a series of cryptophycin compounds that can be useful for disrupting the microtubulin system, as antineoplastic agents, as antifungals, and for the treatment of cancer.
WO 97/08334 reports novel cryptophycin compositions that exhibit a broad spectrum of antineoplastic activity similar to presently used antineoplastic agents such as vinblastine, taxol, and adriamycin.
WO 97/23211, WO 97/31632, WO 98/08506, WO 98/08505, and WO 98/08829 report novel cryptophycin compounds which can be useful for disrupting the microtubulin systems, as antineoplastic agents, and for the treatment of cancer.
Cryptophycin molecules are known to be somewhat toxic. Accordingly, it would be desirable to have cryptophycin molecules with lower toxicity that nonetheless retain the anti-cancer activity of the original cryptophycin molecules. Additionally, cryptophycin compounds having even greater solubility and stability are desired for most pharmaceutical uses. Surprisingly, applicants have now discovered the compound of this invention provides remarkably greater aqueous solubility as well as a superior ability to disrupt the microtubule system to any other compounds disclosed in WO 98/08505. This compound also has a lower toxicity than other cryptophycin molecules. Because of the lower toxicity, higher dosing levels may be achieved for a higher degree of cancer treatment.
As noted in WO 98/08505, such compounds can be prepared using total synthetic methods and are therefore well suited for development as pharmaceutically useful agents.
The present invention provides a compound of Formula I: 
or a pharmaceutically acceptable salt thereof.
This invention also provides a pharmaceutical formulation that comprises, in association with a pharmaceutically acceptable carrier, diluent or excipient, a compound of Formula I.
The present invention provides a method for the treatment of hyperproliferative conditions such as cancer, neoplasm, and hyperproliferative cell growth in mammals comprising administering to a mammal in need of such treatment a pharmaceutically effective amount of the compound of Formula I or a pharmaceutically acceptable salt thereof.
Furthermore, the present invention provides a method for disrupting a microtubulin system in a mammal comprising administering to a mammal in need of such treatment an effective amount of a compound of Formula I.
This invention also provides the use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of hyperproliferative conditions such as cancer, neoplasm, and hyperproliferative cell growth in mammals. Additionally, this invention provides a pharmaceutical formulation adapted for the treatment of hyperproliferative conditions such as cancer, neoplasm, and hyperproliferative cell growth containing the compound of Formula I, or a pharmaceutically acceptable salt thereof.
This invention further provides the use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for disrupting a microtubulin system. Additionally, this invention provides a pharmaceutical formulation adapted for disrupting a microtubulin system containing the compound of Formula I or a pharmaceutically acceptable salt thereof.
The compound of Formula I is also known as Cryptophycin-309. We have found that Cryptophycin-309 exhibits lower toxicity than other cryptophycins but retains the anti-cancer activity of previously known cryptophycin molecules. Because of the lower toxicity, higher dosing levels may be achieved allowing for a higher degree of cancer treatment.
As used herein, the term xe2x80x9cmammalxe2x80x9d shall refer to the Mammalia class of higher vertebrates and includes, but is not limited to, a human. The term xe2x80x9ctreatingxe2x80x9d as used herein includes prophylaxis of the named condition or amelioration or elimination of the condition once it has been established. The compound of Formula I claimed herein can be useful for veterinary health purposes as well as for the treatment of a human patient.
The present invention provides methods for disrupting a microtubulin system in a mammal and for the treatment of hyperproliferative conditions such as cancer, neoplasm, and hyperproliferative cell growth in mammals by administering to a mammal in need of such treatment a pharmaceutically effective amount of a compound of Formula I. The preferred mammal is human.
Because the compound of Formula I is an amine, it is basic in nature and accordingly reacts with any of a number of inorganic and organic acids to form pharmaceutically acceptable acid addition salts. It is preferable to convert the free amine to a pharmaceutically acceptable acid addition salt for ease of handling and administration. Acids commonly employed to form such salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids, such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid and the like. Examples of such pharmaceutically acceptable salts thus are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen-phosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxy-benzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, xcex1-hydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and the like. A preferred pharmaceutically acceptable salt is the hydrochloride salt.
Cryptophycin-309, the compound of Formula I, may be prepared from Cryptophycin-292 by methods well known to the skilled artisan as illustrated in Scheme I: 
The transformation of the epoxide to the chlorohydrin is accomplished generally by the procedure described in U.S. Pat. No. 6,252,064, incorporated herein by reference. Briefly, the epoxide, Cryptophycin-292, is treated with trimethylsilyl chloride in a suitable solvent, typically chloroform, at reduced temperature, typically about xe2x88x9263xc2x0 C., to provide, after silica gel chromatography, the chlorohydrin Cryptophycin-296. This resulting chlorohydrin is then coupled under standard conditions with N-(tert-butoxycarbonyl)glycine to form the corresponding ester. The N-(tert-butoxycarbonyl) substituent is removed by treatment with a suitable acid, typically hydrogen chloride or hydrochloric acid, to provide the compound of Formula I.
Cryptophycin-292 may be prepared by general methods for synthesizing cryptophycin molecules well known in the art. Cryptophycin molecules have typically been synthesized by a convergent synthesis in which four (4) subunits are assembled. The combined AB-subunit has the following formula: 
The preparation of this AB-subunit is well known in the art (See: WO 98/09955, WO 98/08505, and WO 00/23429).
The C-subunit, xcex2-alanine, has the following formula: 
and is commercially available in a protected form, or may be prepared as described by Barrow, et al. (Journal of the American Chemical Society, 117(9), 2479-2490 (1995)) by White, et al., (Journal of Organic Chemistry, 64(7), 6206-6216 (1999)), or in EP 1 059 286 A1.
The D-subunit, 2(S)-(xe2x88x92)-4,4-dimethyl-2-hydroxypentanoic acid, has the following formula: 
and may be prepared as described in the following scheme. 
Briefly, a solution of L-neopentylglycine (a) in sulfuric acid is treated with sodium nitrite to provide the compound of Formula IV.
Alternatively, a protected form of the compound of Formula IV may be prepared as described in Scheme III. 
Tert-butylacetic acid (b) is reduced with a hydride reducing agent, preferably lithium aluminum hydride, to provide 3,3-dimethyl-1-butanol (c). Oxidation of alcohol (c) with 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TEMPO), provides 3,3-dimethylbutyraldehyde (d), which is isolated by co-distillation with tetrachloroethane. Aldehyde (d) is treated sequentially with trimethylsilyl cyanide and a catalytic amount of zinc iodide, followed by methanol to afford (xc2x1)-4,4-dimethyl-2-hydroxypentanenitrile (e). This nitrile is hydrolyzed first with hydrochloric acid, followed by 50% NaOH to provide racemic pentanoic acid (f). Sequential treatment of racemate (f) with neat acetyl chloride, followed by either oxalyl chloride or thionyl chloride and N,N-dimethylformamide, provides 4,4-dimethyl-2-acetoxypentanoyl chloride (g), which is then coupled with (R)-(xe2x88x92)-4-phenyl-2-oxazolidinone in the presence of 4-dimethylaminopyridine and N,N-diisopropylethylamine in dichloromethane to give a mixture of the acetoxy diastereomers. This diastereomeric mixture of acetates is separated by column chromatography to provide the single diastereomeric acetate (h). The acetate (h) is treated with sodium hydroxide in a solution of tetrahydrofuran/methanol/water to give the desired (2S)-(xe2x88x92)-4,4-dimethyl-2-hydroxypentanoic acid IV. The crude pentanoic acid IV is converted to an ester, preferably the allyl ester, and is then purified by chromatography to provide allyl (2S)-(xe2x88x92)-4,4-dimethyl-2-hydroxypentanoate (i).
Cryptophycin-292 is assembled from these subunits as illustrated in the following scheme. 
Coupling of allyl ester (i) with the N-tert-butoxycarbonyl derivative of the C-subunit is accomplished by reaction of the two components in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine in dichloromethane, providing the allyl ester of the CD-subunit (j). The allyl protecting group is removed with morpholine in the presence of tetrakis(triphenylphosphine)palladium(O) in tetrahydrofuran furan to afford the CD-subunit (k), which is then coupled with the AB-subunit (II) in the presence of dicyclohexylcarbodiimide and dimethylaminopyridine in dichloromethane to provide Seco-289. Deprotection of Seco-289 is accomplished by sequential treatment with trifluoroacetic acid and K2CO3. Cyclization of Seco-289 is accomplished by reaction with 2-hydroxypyridine in toluene to provide Cryptophycin-289. Epoxide formation is performed by reaction with 3-chloroperoxybenzoic acid at room temperature to provide Cryptophycin-292.
To a solution of L-neopentylglycine (2.9 gm, 20 mMol) in 1.0 N sulfuric acid (55 mL) is added slowly sodium nitrite (2.76 gm, 40 mMol) at 0xc2x0 C. The mixture is stirred vigorously overnight and is allowed to warm to room temperature. Ethyl acetate (25 mL) is added and the phases are separated. The aqueous phase is extracted with ethyl acetate (2xc3x9780 mL). The combined organic phases are washed with 0.5 N hydrochloric acid, dried, and concentrated to provide 2.3 gm (79%) of the desired compound as a colorless wax. The aqueous phase was treated with additional 1.0 N sulfuric acid (8 mL) and sodium nitrite (2.76 gm) to provide an additional 0.5 gm of the title compound.
[xcex1]D (CHCl3)=xe2x88x926.0xc2x0 (c=5.0)
1H-NMR (300 MHz, CDCl3): xcex44.33 (dd, J=9.5, 2.2, 2H), 1.81 (dd, J=2.4, 14.6, 3H), 1.53 (dd, J=9.3, 14.6, 2H), 1.02 (s, 5H).
A. 3,3-Dimethyl-1-butanol (c)
A mechanically stirred suspension of lithium aluminum hydride (33.5 g, 0.883 mol) is prepared in diethyl ether (1 L, anhydrous) under nitrogen and cooled to 0xc2x0 C. To this suspension is added tert-butylacetic acid (b) in diethyl ether (500 mL) dropwise, and at such a rate as to keep the contents in the flask (3 hours). After complete addition, the ice bath is removed and the suspension stirred overnight at room temperature. The mixture is again cooled to 0xc2x0 C. and the reaction quenched by the slow addition of water (200 mL), followed by hydrochloric acid (12 N, 100 mL). The organic layer is decanted away from the aluminum salts and the salts are washed with diethyl ether (500 mL). The combined organic layers are washed with hydrochloric acid (1 N, 500 mL), sodium bicarbonate (saturated aqueous, 500 mL), sodium chloride (saturated aqueous, 500 mL), dried (magnesium sulfate), filtered, and the filtrate evaporated (40xc2x0 C., 20 mm Hg). The residue is distilled through a 140 mm Vigreaux column (bp 140-150xc2x0 C., 760 mm Hg under N2) to provide the desired compound as a colorless oil, 74.6 g (85%).
1H NMR (CDCl3) xcex43.73 (t, J=7.6 Hz, 2H), 1.55 (t, J=7.6 Hz, 2H), 1.3 (bs, 1H), 0.96 (s, 9H).
B. (xc2x1)-4,4-Dimethyl-2-hydroxypentanenitrile (e)
A mechanically stirred solution of 3,3-dimethyl-1-butanol (c) (74.6 g, 0.730 mol) is prepared in 1,1,2,2-tetrachloroethane (400 mL) and water (2.5 L). The partition is stirred vigorously and treated sequentially with sodium bicarbonate (111 g, 1.32 mol), potassium carbonate (18.5 g, 0.134 mol), TEMPO (5.00 g, 0.032 mol), tetrabutylammonium chloride (20.1 g, 0.0723 mol), and N-chlorosuccinimide (110.7 g, 0.829 mol). After stirring overnight at room temperature, the layers are separated and the organic layer washed with hydrochloric acid (1 N, 200 mL), sodium bicarbonate (saturated aqueous, 200 mL), sodium chloride (saturated aqueous, 200 mL), dried (magnesium sulfate), and filtered. The filtrate is distilled through a 140 mm Vigreaux column (b.p 140-150xc2x0 C., 760 mm Hg under N2) to provide a solution of 3,3-dimethylbutyraldehyde (d) in 1,1,2,2-tetrachloroethane (616 g total weight). 1H NMR shows the solution to be about 8.9% by weight aldehyde, approximately 55 g (77%). The solution is cooled to 0xc2x0 C., stirred, and treated sequentially with zinc iodide (3.00 g, 9.40 mmol), and trimethylsilyl cyanide (83.0 mL, 0.622 mol) dropwise, over a 30 minute period. The ice bath is removed and the solution is stirred at room temperature for 2 hours. Methanol (50 mL, 1.23 mol) is added dropwise (10 min) and stirring is continued for 1 hour. The solution is washed once with water, then evaporated (40xc2x0 C., 20 mm Hg, two 100 mL portions of water are added to azeotrope with 1,1,2,2-tetrachloroethane during evaporation) to provide 70 g (xc2x1)-4,4-dimethyl-2-hydroxypentanenitrile (e) as a reddish oil (75%).
1H NMR (CDCl3) xcex44.58 (t, J=1.2 Hz, 1H), 2.5 (bs, 1H), 1.9 (m, 2H), 1.05 (s, 9H).
C. (xc2x1)-4,4-Dimethyl-2-hydroxypentanoic Acid (f)
A solution of 4,4-dimethyl-2-hydroxypentanenitrile (f) (70.0 g, 0.550 mol) is prepared in hydrochloric acid (12 N, 150 mL, 1.80 mol, the solution immediately refluxes) and is stirred overnight at room temperature. The resulting thick slurry is diluted with diethyl ether (500 mL) and 1:1 saturated aqueous sodium chloride/water (500 mL). The layers are separated and the aqueous layer extracted with diethyl ether (200 mL). The combined organic layers are evaporated (40xc2x0 C., 20 mm Hg) and the residue stirred in water (200 mL), treated with sodium hydroxide (50% solution in water, 50.0 g, 0.625 mol), and heated to 80xc2x0 C. for 2 h. The heat is removed and the solution poured into cracked ice (ca. 500 g) containing hydrochloric acid (12 N, 70 mL, 0.84 mol). This aqueous suspension is extracted with diethyl ether (2xc3x97500 mL), and the combined extracts are dried (magnesium sulfate), filtered, and the filtrate evaporated (40xc2x0 C., 20 mm Hg) to provide 67 g (83%) (xc2x1)-4,4-dimethyl-2-hydroxypentanoic acid (f) as a colorless solid.
mp=72-73xc2x0 C.
MS(FIA): m/e (% relative intensity) 145.1 (M+xe2x88x921, 100).
EA: Calculated C7H14O3 (146.18): C, 57.51; H, 9.65. Found: C, 57.75; H, 9.34.
D. (4R,2xe2x80x2S)-(xe2x88x92)-3-(4xe2x80x2,4xe2x80x2-Dimethyl-2xe2x80x2-acetoxypentanoyl)-4-phenyloxazolindin-2-one (h)
A solution of (xc2x1)-4,4-dimethyl-2-hydroxypentanoic acid (f) (40.0 g, 0.274 mol) is prepared in acetyl chloride (70.0 mL, 0.984 mol) and stirred at room temperature for 2 hours. The solution is concentrated (40xc2x0 C., 20 mm Hg), the residue dissolved in dichloromethane (300 mL), and treated sequentially with oxalyl chloride (30.0 mL, 0.344 mol) and dimethylformamide (0.2 mL, 3 mmol). After stirring overnight at room temperature, the solution is concentrated (40xc2x0 C., 20 mm Hg), stirred in dichloromethane (400 mL), and treated with N,N-diisopropylethylamine (120 mL, 0.689 mol), dimethylaminopyridine (0.50 g, 4.1 mmol), and (R)-(xe2x88x92)-4-phenyl-2-oxazolidinone (50.0 g, 0.306 mol), resulting in a gentle reflux. After stirring overnight at room temperature, the mixture is washed with hydrochloric acid (1 N, 2xc3x97500 mL). The aqueous layer is extracted with dichloromethane (100 mL) and the organic layers are washed with sodium chloride (saturated aqueous, 200 mL), dried (magnesium sulfate), filtered, and the filtrate evaporated (40xc2x0 C., 20 mm Hg) to provide 100 g of the crude diastereomers as a paste. The paste is divided into two 50 g portions and chromatographed on silica gel [1 kg, 100 mm column, 4:1:1 tert-butyl methyl ether/dichloromethane/hexanes] to provide 42.5 g of oxizolidinone (h) as a colorless solid (47%). An analytical sample is prepared by recrystallizing 0.78 g from 40 mL of 1:1 tBuOMe/hexanes to afford 0.61 g of pure (h) as white needles.
mp=148-149xc2x0 C.
[xcex1]D25=xe2x88x92123.6xc2x0 (c=1.03, CHCl3)
MS(FIA): m/e (% relative intensity) 351.0 (50), 334.2 (M++1, 70), 274.0 (100).
EA: Calculated. for C18H23NO5 (333.38): C, 64.85; H, 6.95; N, 4.20. Found: C, 65.08; H, 7.04; N, 4.28.
The column above provides 40.0 g of a white solid, which is recrystallized from 5:2 tBuOMe/hexanes (350 mL total) to afford 30.0 g (33%) of the opposite isomer of oxizolidinone (h) as fine, white needles.
mp=140-141xc2x0 C.
[xcex1]D25=xe2x88x9280.50xc2x0 (c=1.00, CHCl3)
MS(FIA): m/e (% relative intensity) 392.2 (20), 351.5 (30), 334.1 (M++1, 50), 274.3 (100).
EA: Calculated for C18H23NO5 (333.38): C, 64.85; H, 6.95; N, 4.20. Found: C, 65.15; H, 6.97; N, 4.43.
E. Allyl (2S)-(xe2x88x92)-4,4-Dimethyl-2-hydroxypentanoate (i)
A solution of (4R,2xe2x80x2S)-(xe2x88x92)-3-(4xe2x80x2,4xe2x80x2-dimethyl-2xe2x80x2-acetoxypentanoyl)-4-phenyloxazolindin-2-one (h) (42.5 g, 0.127 mol) is prepared in tetrahydrofuran (750 mL), CH3OH (350 mL), and water (350 mL) and treated with sodium hydroxide (25 g of a 50% solution in water, 0.313 mol). After stirring overnight at rt, the mixture is concentrated (20xc2x0 C., 20 mm Hg) to about xc2xc volume, diluted with diethyl ether (500 mL), treated with hydrochloric acid (12 N, 30 mL, 0.36 mol) and sodium chloride (50 g), and stirred for 15 minutes. The layers are separated and the aqueous layer extracted with diethyl ether (2xc3x97500 mL). The organic layers are concentrated (40xc2x0 C., 20 mm Hg) and the residue stirred in water (250 mL) and treated with sodium bicarbonate (20.0 g, 0.200 mol) and tetrabutylammonium bromide (46.0 g, 0.142 mol). The mixture was heated to 45xc2x0 C., stirred, and treated with allyl bromide (31.4 g, 0.260 mol), neat, over a 6 hour period. Stirring is continued at 45xc2x0 C. for 2 hours after complete addition and then at room temperature overnight. Hexanes (200 mL) are added and the mixture is made strongly acidic with hydrochloric acid (3 N, 100 mL). The layers are separated and the aqueous layer extracted with hexanes (200 mL). The organic layers are washed with hydrochloric acid (1 N, 100 mL), sodium chloride (saturated aqueous, 200 mL), dried (magnesium sulfate), filtered, and the filtrate evaporated (40xc2x0 C., 20 mm Hg) to provide 16.8 g of an amber oil. The oil is chromatographed on SiO2 (70xc3x97160 mm column, 15% tBuOMe in hexanes) to provide 14.4 g of (i) (61%).
[xcex1]D25=xe2x88x926.320 (c=1.17, CHCl3)
MS (FD+) m/e (% relative intensity) 186.1 (M+, 100).
A. Coupling
A solution of (R)-3-[(tert-butoxycarbonyl)amino]-2-methylpropanoic acid (16.0 g, 0.789 mol) and allyl (2S)-(xe2x88x92)-4,4-dimethyl-2-hydroxypentanoate (i) (14.0 g, 0.0752 mol) is prepared in dichloromethane (100 mL, anhydrous) under nitrogen and treated with dimethylaminopyridine (0.50 g, 4.1 mmol). The solution is stirred, cooled to 0xc2x0 C., and treated with dicyclohexylcarbodiimide (22.1 g, 0.107 mol) in dichloromethane (100 mL, anhydrous) dropwise and at such a rate as to keep the temperature at or below +10xc2x0 C. The ice bath is removed and the mixture stirred for 4 hours. The mixture is filtered, concentrated (20xc2x0 C., 20 mm Hg), and the residue chromatographed on silica gel (1 kg, 100 mm diameter column, 15% tBuOMe in hexanes) to provide 28.8 g of allyl (2S,2xe2x80x2R)-(xe2x88x92)-2-[3xe2x80x2-[[(tert-butoxycarbonyl)amino]-2xe2x80x2-methylpropanoyl]oxy]-4,4-dimethylpentanoate (j) as a colorless oil in quantitative yield.
[xcex1]D25=xe2x88x9245.4xc2x0 (c=0.99, CHCl3)
MS(FIA): m/e (% relative intensity) 372.3 (M++1, 37), 316.1 (25), 272.4 (100).
B. Deesterification
A solution of pentanoate (j) (24.4 g, 65.7 mmol) is prepared in tetrahydrofuran (300 mL, anhydrous) and stirred under nitrogen. The solution is treated sequentially with morpholine (65.0 g, 0.746 mol) and tetrakis(triphenylphosphine) palladium(O) (320 mg, 0.276 mmol), then stirred for 3.5 hours. The mixture is diluted with diethyl ether (500 mL) and washed with hydrochloric acid (250 mL of 3 N, followed by 250 mL of 1 N). The aqueous layers are extracted with diethyl ether (200 mL), and the combined organic layers are washed with sodium chloride (saturated aqueous, 200 mL), dried (magnesium sulfate), filtered, and the filtrate evaporated (40xc2x0 C., 20 mm Hg) to provide 22.2 g of the title compound as a yellow paste in quantitative yield.
[xcex1]D25=xe2x88x9240.9xc2x0 (c=1.09, CHCl3)
MS(FIA): m/e (% relative intensity) 332.2 (M++1, 10), 276.2 (15), 232.2 (100).
A solution of (R)-3-[(tert-butoxycarbonyl)amino]-2-methylpropanoic acid N-hydroxysuccinimide ester (0.30 g, 1.0 mmol) and (2S)-(xe2x88x92)-4,4-dimethyl-2-hydroxypentanoic acid (IV) (0.219 g, 1.5 mmol) is prepared in acetonitrile (10 mL, anhydrous) and treated with dimethylaminopyridine (0.525 g, 4.3 mmol). After stirring for 18 hours, the acetonitrile is removed by evaporation. The residue is treated with hydrochloric acid (0.1 M) and the resulting mixture is extracted with ethyl acetate (2xc3x9750 mL). The organic phases are combined, dried, and concentrated. The residue is chromatographed (C18 reversed-phase column, YMC ODS gel), eluting with 1:1 methanol:water followed by methanol to give 0.268 gm (81%) of the title compound as a colorless wax.
[xcex1]D25=xe2x88x9237.5xc2x0 (c=1.4, CHCl3)
MS(FAB): m/e (% relative intensity) 354 (M++Na, 100), 298 (11.8), 276 (7.2), 232 (9.6), 130 (8.1).
MS(HRFAB): m/e=354.1878 (Calculated for C16H29NO6: 354.1893).